ABSTRACT

Giardia and Cryptosporidium are pathogenic protozoa often present in the environment in their infective form(cysts and oocysts). These parasites are very resistant to disinfection, which makes them important target organisms in environmental quality monitoring and sanitation. Viability assessment provides an interpretation of cell inactivation, and it can be evaluated by membrane integrity as well as enzyme activity, using different staining methods. These are straightforward and adequate to laboratories that lack infrastructure for molecular-based technologies or animal infectivity tests. This study investigated simultaneous staining by a commercial live/dead kit, in order to assess viability of Cryptosporidium parvum oocysts and Giardia muris cysts, comparing it to propidium iodide (PI) incorporation, a common stain applied in viability estimation. Results suggested that, although the central hypothesis of one-panel visualization (α = 0.05) was met, simultaneous staining impaired (oo)cyst detection by immunofluorescence assay (IFA), which was found to be essential to enumeration, as the live/dead test led to poor (oo)cyst labelling or a 10-fold lower recovery when carried out concomitantly to IFA. As for the viability assessment itself, although red dye uptake occurred as expected by dead or weakened organisms, neither live G. muris cysts or C. parvum oocysts present any green fluorescence by esterase metabolism. This may have been caused by low enzyme activity in the infective form and/or wall thickness of these parasites. The results do not exclude the possibility of simultaneous fluorescence staining for protozoa, but it is a starting point for a broader analysis, that may consider, for instance, different incubation conditions.

摘要

贾第鞭毛虫和隐孢子虫是致病性原生动物，通常以其感染形式（囊肿和卵囊）存在于环境中。这些寄生虫对消毒有很强的抵抗力，这使其成为环境质量监测和卫生的重要目标生物。活力评估提供了对细胞失活的解释，并且可以使用不同的染色方法通过膜完整性和酶活性进行评估。这些对于缺乏分子技术或动物传染性测试基础设施的实验室来说是直接且足够的。本研究通过商业活/死试剂盒调查同时染色，以评估隐孢子虫卵囊和鼠贾第鞭毛虫的生存能力囊肿，将其与碘化丙啶 (PI) 掺入进行比较，这是一种用于生存力估计的常见染色剂。结果表明，尽管满足了单面板可视化的中心假设（α  = 0.05），但同时染色损害了免疫荧光测定法（IFA）对（OO）囊肿的检测，这对计数至关重要，因为活/死当与 IFA 同时进行时，测试导致（oo）囊肿标记不良或恢复率降低 10 倍。至于生存能力评估本身，虽然死亡或减弱的生物体如预期的那样发生了红色染料的吸收，但活的G. muris囊肿或C. parvum都没有卵囊通过酯酶代谢呈现任何绿色荧光。这可能是由于这些寄生虫的感染形式和/或壁厚的低酶活性引起的。结果并不排除同时对原生动物进行荧光染色的可能性，但它是更广泛分析的起点，可以考虑例如不同的孵育条件。